Cytokinesis, the physical process that divides daughter cells, is of fundamental importance to development and growth regulation. In many instances, cytokinesis is coupled with the asymmetric segregation of cellular determinants, which in turn results in the functional diversification of cell types. During the systematic identification of maternal-effect mutations in the zebrafish, two mutations, in the genes aura and nebel, were identified which affect both of these processes. Mutations in these genes result in embryonic lethality due to defects in cell adhesion and membrane deposition. In both cases, the basis of this phenotype is a defect in the completion of cytokinesis, possibly in the addition or stabilization of new membrane at the cleavage plane. In addition, both of these mutations affect different steps in a pathway of segregation of the vasa RNA, a component of a specialized cytoplasm that confers the germ cell fate. Both types of defects may be based on the inability of the cytoskeleton to undergo specific rearrangements required for its function: aura-dependent microfilament rearrangements at multiple stages during early cleavage and nebel-dependent microtubule reorganization at the forming furrow. The proposed research will determine the precise roles of these genes in the process of cytokinesis and the segregation of the vasa RNA. For this purpose we will use cell biological techniques to visualize cellular components in wild-type and mutant embryos and inhibitor studies to determine the role of subcellular processes or networks. In addition, we will determine the molecular identity of the affected genes through a positional cloning approach. The understanding of these processes will provide insights on a variety of poorly understood subjects: i) the processes involved in the completion of cytokinesis, ii) the asymmetric segregation of RNA and other products within dividing cells, and iii) the functional relationship between the cellular mechanisms required for these two processes.